Project Summary High-risk neuroblastoma (HR NB) is a lethal pediatric solid tumor. Survival is < 50% and those that survive suffer many treatment related toxicities, stressing a critical need for novel tumor- targeted therapies. NB patient survival improved with the addition of the anti-GD2 antibody, dinutuximab, increasing the interest in other immunotherapies like adoptive transfer of cellular therapies for NB. Cellular therapy has so far focused on ?? T cells and NK cells, which to date have been uniformly unsuccessful for solid tumors. Gamma delta (??) T cells are an innovative and superior choice as they are MHC independent, directly cytotoxic to tumor cells, including NB, can recognize and target immune-suppressing cells in the tumor microenvironment, and lack the alloreactivity of ?? T cells. Adoptive ?? T cells have not been widely used clinically to date due to inadequate ex vivo expansion methods. We have developed a GMP-compliant serum free manufacturing strategy to expand ?? T cells to sufficient levels for human use. Expanded cells highly express CD16, which is directly involved in antibody directed cellular cytotoxicity (ADCC). We have shown that ?? T cells expanded from NB patients effectively kill NB cell lines and enhance dinutuximab-induced NB cell death. Therefore, our primary objective is to generate preclinical and IND enabling data demonstrating the effectiveness of our ?? T cell product. We have also generated two chimeric antigen receptors (CARs) targeting GD2 to localize ?? T cells to NB, and show that CAR-modified immunocompetent cells have enhanced cytotoxicity compared to non-modified cells. We have also developed strategies to confer chemotherapy resistance to normal immune cells. This led to our innovative ?drug resistant immunotherapy? platform whereby chemo-protected ?? T cells can be co-administered with chemotherapy to significantly augment anti-NB efficacy. Our second objective is therefore to genetically engineer ?? T cells using a novel recombinant viral vector-based delivery of cDNA sequences that encode for methylguanine methyltransferase, MGMT, a temozolomide (TMZ) resistance gene and our anti-GD2-CAR. Modified ?? T cells will be analyzed for various genetic and functional characteristics and anti-tumor potency, both alone and with TMZ, using NB cell lines and patient derived xenografts both in vitro and in vivo. The goals of this proposal are to 1) develop robust preclinical data to support the first in pediatrics use of autologous expanded ?? T cells for recurrent NB, and 2) determine whether drug resistant/GD2CAR ?? T cells are superior to unmodified ?? T cells to inform future clinical studies. Success of this immunotherapy platform in NB will provide the foundation to treat other cancers using a similar strategy.